Use of lipo chitooligosaccharides to initiate early flowering and fruit development in plants and related methods and compositions

ABSTRACT

The present invention relates to the use of LCOs in initiating earlier flowering, increased number of buds and flower buds and earlier fruit development in non legume and legume plants, as compared to flowering and fruit development under conditions without use of LCOs, and the enhancement of plant growth and yield associated therewith; to compositions comprising an effective amount of at least one LCO and agriculturally acceptable carriers, associated with earlier flowering, increased bud and flower numbers and earlier initiation of fruit development as compared to conditions without use of LCOs, and with increased growth and plant yield; and to methods using LCOs and compositions of one or more LCOs and agriculturally acceptable carriers, associated with earlier flowering initiation, increased bud and flower numbers and earlier fruit development in both legume and non-legume crop plants as compared to conditions without use of LCOs and associated enhancement of growth and yield.

FIELD OF THE INVENTION

The present invention relates generally to the fields of horticultureincluding but not limited to flowers, fruits, vegetables, nuts,turfgrass, herbs, spices, ornamental shrubs and trees, aquatic plantsand mushrooms grown outdoors or in greenhouses or indoors for bothcommercial or personal use and agriculture and more specifically to theuse of Lipo-chitooligosaccharides (LCOs) and compositions thereof toinduce early flowering, increase the number of buds and flowers,initiate earlier fruiting, earlier maturity and increase yields inplants and to methods of inducing earlier flowering and initiation ofearlier fruiting in plants by exposure to LCOs and compositions of same.

BACKGROUND OF THE INVENTION

There is a growing interest in the role of LCOs and compositions thereoffor enhancement of plant seed germination, seedling emergence and growthof plants both for crop and horticultural purposes in both legumes andnon-legumes. Compositions for accelerating seed germination and plantgrowth are provided in Application No. PCT/CA99/00666, published Feb. 3,2000, WO 00/04778, all contents of which are incorporated herein byreference. There is also an interest in the possible effects of LCOs inplant photosynthesis and PCT/CA00/01192, published Apr. 19, 2001, WO01/26465 A1 describes the use of LCOs and compositions of LCOs forincreasing plant photosynthesis. Chemical structures of LCOs aredescribed in U.S. Pat. Nos. 5,175,149; 5,321,011 and 5,549,718.Synthetic LCOs are also known.

There is great interest in the field of agricultural research,particularly in the field of plant growth promoters, of plantphysiological processes which may be affected by LCOs. Prithiviraj etal, Planta (2003) 216:437-445 discuss certain observed inducedphysiological changes in both host and non-host plants by LCOs, allcontents of which is incorporated herein by reference.

LCOs are known to be released by Rhizobia, symbiotic bacteria primarilyof the genera Generarhizobium, Bradyrhizobium, Sinorhizobium,Mesorhizobium and Azorhizobium and the like, the Rhizobiacese familybeing in a state of taxonomic flux Both of the aforesaid Internationalapplications summarize current understanding of the specializedsymbiotic relationship of Rhizobia with legume host plants in theformation of nodule organs and associated fixation of atmosphericnitrogen within these organs, as well as the plant to bacteria signaland bacteria to plant signal interaction associated with such symbioticrelationship.

Although there is a considerable body of knowledge on the influence ofLCOs on typical host plant physiology, there is a growing interest inthe effect of LCOs on plant growth with respect to both host andnon-host plants, particularly by application of the molecule withoutnecessarily the fostering of a micro-organism and plants symbioticrelationship.

The body of knowledge relating to the possible role of LCOs, in bothhost and non-host plants and on processes associated with plant growthpromotion continues to grow, with particular practical interest in theeffects of LCOs on plant physiology and processes relevant to increaseplant yields, not only with respect to commonly considered crop plants,both host and non-host, but also with respect to horticulture species.

Thus, there continues to be a need to study the effects of LCOs on plantgrowth, in addition to processes relating to nodulation and nitrogenfixation in legume host plants and to germination, emergence andphotostimulation in both legume and non-legume plants. In particular,there is a need to study the effect of LCOs on bud and floweringinitiation, budding, fruiting initiation and development, generally inrelation to growth and maturity of plants, both leguminous andnon-leguminous and the affect on plant yields. The present inventionendeavours to address these and other needs.

SUMMARY OF THE INVENTION

The present invention relates to the use of LCOs in initiating earlyflowering and budding, increased flowering and budding and earlier fruitdevelopment in non-legume and legume plants, as compared to floweringand fruit development under conditions without use of LCOs, and theenhancement of plant growth and yield associated therewith. The presentinvention also relates to agricultural compositions comprising aneffective amount of at least one LCO and agriculturally acceptablecarriers, associated with early flowering and budding, increasedflowering and budding, earlier plant maturity and earlier initiation offruit development as compared to conditions without use of LCOs, andwith increased growth and plant yield. The present invention furtherrelates to methods using LCOs and compositions of one or more LCOs andagriculturally acceptable carriers, associated with earlier floweringinitiation and budding, increased flowering and budding and earlierplant maturity and earlier fruit development in both legume andnon-legume crop plants as compared to conditions without use of LCOs andotherwise associated enhancement of growth and yield, and all asexemplified herein below.

Surprisingly, the compositions of the present invention affect not onlylegume varieties but also a wide and divergent variety of non-legumeplants, including crop plants and horticultural and bedding plantspecies in the initiation of earlier flowering and budding, increasedflowering and budding, earlier maturity and earlier fruit development,and increased yield, as compared to conditions where LCOs are notapplied and all as exemplified herein below.

According to the present invention, in both legume and non-legumeplants, the administration of an effective amount of LCO or LCOs, or ofcompositons of one or more LCOs with agriculturally suitable carriers,initiates buddy and/or flowering at an earlier stage, increases totalbud and/or flower numbers and also causes earlier fruit development andplant maturity as compared to conditions without use of LCOs, includingan associated increase in yield. Administration of LCOs for such purposemay be by leaf or stem application, or application in the proximity ofthe seed, root or plant. Such methods are non-limiting and may includeother methods, which would be understood by the skilled person,including by administration of micro-organisms known to release LCOs inthe proximity of a plant seed, or seedling in any stage of emergence, orin the proximity of a plant, including in the vicinity of the root androot hairs. The same would be with respect to application of LCOsindependent of the micro-organisms known to release such molecules.

Thus, in accordance with a further embodiment of the present invention,there is provided a method for the initiation of earlier flowering,increased budding and flowering and earlier fruit development and plantmaturity in non-legume and legume plants associated with the growth andyield of a plant, comprising the treatment of a plant with an effectiveamount of one or more LCOs or a composition comprising an agriculturallyeffective amount of one or more LCOs in association with anagriculturally suitable carrier or carriers, wherein the effectiveamount has the effect of initiating earlier flowering and/or buddingand/or increased bud and/or flower number and/or earlier fruitdevelopment and/or plant growth and/or yield, as compared to anuntreated plant, and all as exemplified herein below. Suitable LCOs foruse according to the present invention include the LCOs as identified inthe aforesaid International applications and patents.

Compositions of the present invention will be understood to include intheir scope, one or more different LCO molecules, as well as comprisingone or more types of molecules other than LCO, including, withoutlimitation, one or more plant to bacteria molecule and/or othermolecules or agents known to promote growth or fitness and mixtures ofsuch compositions.

The inventors and applicant herein are the first to show, as exemplifiedin the greenhouse and field experiments set out hereafter, that acomposition comprising an LCO can have a significant affect on bothlegume and non-legume plants by initiating early bud and/or flowering,increased bud and/or flowering and earlier fruit development and/oryield, as compared to conditions without use of LCOs, and theenhancement of plant maturity, growth and yield associated therewith.Non-limiting examples of crop plants include dicotyledons andmonocotyledons and legumes. From the aforesaid experiments and as setout below, it can be predicted that such results will apply to crop,horticultural and personal use plants, legumes and non-legumes,including, but not limited to, flowers, fruits, vegetables, nuts,tubers, turf grass, herbs, spices, ornamental shrubs and trees, aquaticplants and mushrooms grown in field or greenhouse for agricultural,commercial and personal use. In view of the plants exemplified hereinand the results, the skilled person will appreciate, can adapt theteaching of the present invention to a diversity of plants, both legumeand non-legume, for crop, horticultural and personal use, including butnot limited to, plants of the families: Fabaceae, Brassicaceae,Solonaceae, Chenopodiaceae, Asteraceae, Malvaceae, Cucurbitaceae andPoaceae.

The term “LCO” as used herein, will be understood as reference ingeneral to a Nod factor which is under control of at least onemodulation gene common to rhizobia, that is bacterial strains which areinvolved in a nitrogen fixing symbiotic relationship with a legume, andwhich serve as micro-organism-to-plant phytohormones which induce theformation of nodules in legumes and enable the symbiotic micro-organismsto colorize said plant modules. LCOs are understood to comprisederivatives of an oligosaccharide moiety, including fatty acid condensedat one end thereof. Non-limiting examples of LCOs are described in U.S.Pat. Nos. 5,175,149; 5,321,011 and 5,549,718. The instant invention isdemonstrated in particular with LCOs from Bradyrhizobium japonicum, butit not so limited.

The uses, compositions and methods of the present invention will beunderstood to include initiation of early bud and/or flowering and/orincreased flowering and/or budding and/or earlier fruit developmentand/or enhanced plant maturity and/or plant growth and yield under bothsub optimal or limiting and non-limiting environmental conditionsassociated therewith. Such sub optimal or limiting environmentalconditions include but are not limited to liming or sub optimalconditions of heat, water pH, soil nitrogen concentrations and the like.

An effective amount of LCO will be understood to relate to uses,compositions and methods of the present invention wherein the amount issufficient to manifest statistically significant earlier budding and/orflowering and/or increased flowering and/or budding and/or earlier fruitdevelopment and/or enhanced maturity and/or plant growth and yieldassociated therewith.

By proximity of seed, root or plant will be understood to relate to anylocation of seed, root or plant wherein soluble materials orcompositions of the present invention will be in actual contact withsaid seed, root or plant.

By bud or budding will be understood conditions consistent with stemswelling consisting of overlapping immature leaves or petals. Byflowering will be understood the process or state of producing one ormore flower.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention having been generally described above, the accompanyingfigures will now be referenced in the discussion of a preferredembodiment of the invention, as set out in the examples which follow, inwhich:

FIG. 1 shows the effect of LCO dose and timing on fruit set of Cobratomatoes; (Same data as table 2)

FIG. 2 shows the effect of LCO dose and timing on fruit number of Cobratomatoes; (Same data as table 2)

FIG. 3 shows the effect of LCO dose and timing on flower number of Cobratomatoes; (Same data as table 1)

FIG. 4 shows the effect of LCO dose and timing on flower number of Cobratomatoes; (Same data as table 1)

FIG. 5 shows the effect of LCO dose on number of flower of Cobratomatoes; (Same data as table 3)

FIG. 6 shows the effect of LCO dose on the number of fruit of Cobratomatoes; (Same data as table 4)

FIG. 7 shows the effect of LCO dose on yield of fruit of Cobra tomatoes;(Same data as table 5)

FIG. 8 shows the effect of LCO on tomato plant flowering;

FIG. 9 shows the effect of LCO on induction of flowering in Arabidopsisthaliana;

FIG. 10 shows the effect of LCO on induction of flowering in Arabidopsisthaliana;

FIG. 11 shows the effect of LCO dose on the yield of fruit per plant, intomato plant application;

FIGS. 2-1: LCO foliar application enhanced early flowering and totalflower number in greenhouse tomatoes.

FIGS. 2-2: LCO foliar application enhanced early fruiting and totalfruit number in greenhouse tomatoes.

FIG. 2.3: Effect of LCO application on earlier flowering and number offlowers in Marigolds.

FIG. 2.4: Effect of LCO application on fruit number of strawberries.

FIG. 2.5: Effect of LCO soil application on cherry tomato early fruitnumbers.

FIG. 2.6: LCO application promoted tomato early fruit number.

FIG. 2.7: LCO application promoted tomato early fruit.

FIG. 2.8: Cumulative harvested fruit number from tomato plants when 50ng/plant LCO was applied once at variable growing stages.

FIG. 2.9: Cumulative harvested fruit yield from tomato plants when 50ng/plant LCO was applied once at variable growing stages.

FIG. 2.10: Effect of LCO application on advancement of hot pepper earlyflowering.

FIG. 2.11: Effect of LCO application on advancement of hot pepperfruiting.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following description ofpreferred embodiments, which is non-restrictive, and with reference tothe accompanying figures, which is exemplary and should not beinterpreted as limiting the scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The following experiments are reported herein, conducted to study theeffect of application of LCO on the initiation of flowering and fruitdevelopment of a host plant under both greenhouse and field conditions.

Trials 1 and 2: Effect of LCO on Greenhouse Tomato

Two Experiments on the Application of LCO to Tomatoes Have BeenUndertaken

Trial 1: Cobra (a hybrid cultivar) was used to examine optimumapplication dose between 10 and 100 ng/plant at one or two applicationsand the LCO delivery medium. The levels chosen were the extremes ofbeneficial doses determined previously in tomato field trials. Thedelivery media tested were LCO in water, Apex and centrifuged Apex. Thefirst application of LCO was made 10 days after transplanting. Whenapplied a second time it was 2 weeks after the first application.

The following parameters were tested: leaf number, plant height, numberof cluster, number of flower, number of fruit. Observations were madecontinuously for approximately a two-month period, at one-week intervalsuntil plant growth was limited by pot bound roots.

A statistically significant difference in early yield was noted between50 ng LCO treatment applied twice and the untreated control (see Table5, FIG. 7, Fruit weight). Other levels of application were notsignificantly better in yield than contol. There was statisticallysignificant effect between 50 ng LCO treatment and control on fruitweight of Cobra. 50 ng treatment showed more uniform results indifferent delivery media. The increase of early yield by 50 ng LCOshowed the potential ability of LCO applied as a growth enhancer ontomatoes.

There was no statistical difference between treatments on flower numberand number of fruit set at any time point. However, the doubleapplication of 50 ng LCO per plant provided a numerically higher flowernumber earlier and also delivered the best early yield. Similarly, Therewas no significant difference on number of fruit among LCO treatments.Fruits appeared 48 days after transplanting and 38 days after firstapplication of treatments and 24 days after second application oftreatments. Plants in LCO 10 ng treatment showed slightly higher numberof fruit than other treatments and than control.

Examination of the first graph in Trial 1 (FIG. 5, Table 3) indicates a4-5 day advance of flowering over control and the second graphdemonstrates an 8-9 day advance in early fruiting over control(horizontal separation between treatment lines). Early fruiting mustarise from earlier flowering.

There was no significant difference on number of flowers among LCOtreatments. Flower buds appeared 30 days after transplanting and 20 daysafter first application of treatments. Flowers started to open 40 daysafter transplanting, and 30 days after first application of treatments.The plants in 50 ng LCO treatment had more flowers than other treatmentsand control at 21% and 14% on January 15 and January 22. On the otherdays, the number of flower was similar among treatments and control.

Trial 2: The Cobra variety was used to re-examine optimal doses of LCO.The concentrations tested were 50 ng and 75 ng LCO per plant, appliedonce (2 weeks after transplanting) and twice (4 weeks aftertransplanting). The sample number was increased to 20 plants.

At 50 ng/plant there was a significant difference from control on numberof flowers over the first three observations (Table 1) (see Dose &timing on Flower No. Cobra Trial 2). Later, treatment significance onflowering was lost but this is to be expected because of the floweringcharacteristics of the tomato plant. Examination of the Flower numberdata for Cobra Trial 2, (FIGS. 3 and 4) indicates an advance inflowering of some 3 days for 50 ng treatment attaining same flowernumber as control. On fruit number the 50 ng LCO treatment applied twiceshowed significantly higher numbers over control for the first 4 weeks.The higher fruit number (Table 2) (see histogram for Fruit number CobraTrial 2) arises from earlier flowering. The graph for Dose and Timing onFruit Set Cobra Trial 2 (FIGS. 1 and 2) demonstrates that a 50 ng/plantapplication twice, advances equivalent fruit numbers by 2 weeks overcontrol.

TABLE 1 The Effect of Different LCO Concentrations on Flower Number ofCobra Tomatoes in Greenhouse Studies Flower Flower Flower Flower FlowerFlower Number/ Number/ Number/ Number/ Number/ Number/ Plant Plant PlantPlant Plant Plant Treatments (Feb. 28) (Mar. 7) (Mar. 14) (Mar. 21)(Mar. 28) (April 4) LCO 50 ng once 0.0 b 2.5 ab 7.4 ab 14.55 23.95 32.45LCO 50 ng twice 0.2 a 3.2 a 8.4 a 16.0 25.15 32.2 LCO 75 ng once 0.0 b2.85 ab 7.35 ab 14.2 21.4 28.0 LCO 75 ng twice 0.0 b 3.25 a 7.35 ab 15.423.35 31.0 Surfactant 0.0 b 1.9 b 5.8 b 12.85 21.65 32.0 500 ppm onceSignificance* P = 0.0006 P = 0.03 P = 0.05 P = 0.168 P = 0.156 P = 0.368*There is significant different when P < 0.05 Notes; Seeding: Jan. 6,Transplanting: Feb. 7, First Application: Feb. 21, Second Application:Mar. 7, 2003 (Greenhouse Tomato Cobra)

TABLE 2 The Effect of Different LCO Concentrations on Fruit Number ofCobra Tomatoes in Greenhouse Studies Fruit Fruit Fruit Fruit Number/Number/ Number/ Number/ Plant Plant Plant Plant Treatments (Mar. 14)(Mar. 21) (Mar. 28) (April 4) LCO 50 ng once 0.05 b 0.25 d 0.525 c 1.3 bLCO 50 ng twice 0.75 a 1.8 a 2.025 a 2.5 a LCO 75 ng once 0.45 ab 0.9 bc1.15 bc 1.45 b LCO 75 ng twice 0.6 ab 1.0 b 1.325 ab 1.9 ab Surfactant0.1 b 0.4 cd 0.7 bc 1.4 b 500 ppm once Significance* P = 0.03 P < 0.0001P < 0.0001 P = 0.01 *There is significant different when P < 0.05 Notes:Seeding: Jan. 6, Transplanting: Feb. 7, First Application: Feb. 21,Second Application: Mar. 7, 2003.

Results: Effect of LCO Dose on Greenhouse Tomato (Cobra)

The cobra seedlings were transplanted 32 days after seeding, the firstapplication was 10 days thereafter, the second application was 14 daysafter the first application. The fruits were harvested 6 weeks after thesecond application.

Results: Effect of LCO Dose on Greenhouse Tomato (Cobra)

TABLE 3 Number of Flowers/Plant Date Treatment Dec. 30 Jan. 07 Jan. 15Jan. 22 Jan. 31 Control 0.7 2 4.2 5.8 8.9  10 ng 1.2 2.4 4.4 5.6 8.1  50ng 0.97 2.5 5.2 6.7 8.7 100 ng 0.9 2.2 4.4 6.2 8.7 Mean 0.9425 2.2754.55 6.075 8.6

There was no significant difference on number of flowers among LCOtreatments. Flower buds appeared 30 days after transplanting and 20 daysafter first application of treatments. Flowers started to open 40 daysafter transplanting, and 30 days after the first application oftreatments. The plants with a 50 ng LCO treatment had more flowers thanother treatments and control at 21% and 14% on January 15 and January22.

TABLE 4 Number of Fruit/Plant Date Treatments Jan. 07 Jan. 15 Jan. 22Jan. 31 Control 0.1 1.6 2 2.7  10 ng 0.3 1.8 2.1 2.5  50 ng 0.33 2.2 2.52.9 100 ng 0.37 1.7 2.1 2.6 Mean 0.275 1.825 2.175 2.675 Notes; SeeTable 3 above.

LCO application at all levels advanced early fruit set, three to fourweeks after application. The optimal application was approximately 50 ngper plant.

TABLE 5 Fruit Weight (Gram/Plant) Treatments Base 0 ng 10 ng 50 ng 100ng Water 92.81 96.11 108.99 88.57 Bacterial Carrier 74.14 97.97 103.3266.26 Bacterial Supernatent 61.65 67.2 100.48 109.13 Mean 76.2 87.09104.26 87.99 Notes; See Table 3 above.

There was statistically significant effect between 50 ng LCO treatmentand control on fruit weight of Cobra. 50 ng treatment showed moreuniform result in different delivery medium. The increase of early yieldby 50 ng LCO showed the potential ability of LCO applied as a growthenhancer on tomatoes. Water was the optimal carrier for LCO applicationin this study.

Experiments 1 and 2

Summary of the Experiment: Effect of LCO on Tomato Flowering

Two experiments were performed to investigate effect of LCO on plant'sflowering using greenhouse-grown tomato plants.

In general, LCO induced early flowering in both experiments (FIGS. 8 and9).

Experiment 1: LCO treatment induced flowering in 25% more plants ascompared to control at day I of assessment. This increase was sustainedthorough out experiment, reaching 35% difference at day 4 of assessment.LCO treatment caused a 3 day-shift in time of flowering, i.e., over 80%of LCO treated plants flowered 3 days earlier than control, non-treatedplants. Early flowering will initiate earlier fruit set and subsequentearlier fruit development, which in turn leads to higher yield oftomatoes.

Experiment 2: Initial assessment of tomato flowering confirmed againthat LCO treatment induces early flowering in tomatoes. Initially, thereis a 10% difference between LCO treated plants and control. Thisdifference increased to 20% by day 3 of assessment. Initial dataobtained in this experiment confirms findings from previous one andfurther supports claim that LCO treatment incuses early flowering inplants.

Experiments 3 and 4

Summary of the Experiment: Effect of LCO on Flowering in

Arabidopsis thaliana

Two experiments were performed to investigate effect of LCO on plant'sflowering using experimental model plant Arabidopsis thaliana.

In general, LCO induced early flowering in both experiments (FIGS. 9 and10).

Plants were treated with various a range of LCO concentrations. It wasfound that treatment with 10⁻⁷ Molar the most effective in induction offlowering. The LCO treated plants reached over 80% of plants with openflowers 4 days earlier than control, surfactant treated plants. LCOinduced faster and more uniform flowering.

Experiment 5: Foliar Application of LCO to Bedding Plants

This is a growth room study. Seeds of garden plant species were selectedon the basis of seed-purchase popularity (Norseco, Montreal), grown intrays of Pro-Mix (NB. trademarked name) Seeding Medium, and at some sizewere transplanted into trays of 36 and 32 wells containing the samemedium. Growth proceeded under lights in the growth room.

Some 2 weeks before expected flowering, 16 young plants were sprayedwith each of various levels of LCO leaving control plants untreated. Theresults are presented in Table 5A on bud formation and where possibleopened flowers.

TABLE 5A Effect of LCO Application on ornamental plants Total Number ofTotal Number of Treatments Impatiens buds Marigold buds UntreatedControl 68 26 buds and 1 flower 20 ml/16 plants of 71 26 buds and 0flower 10⁻⁷ M LCO application 50 ml/16 plants of 66 34 buds and 3flowers 3 × 10⁻⁸ M LCO application 20 ml/16 plants of 85 24 buds and 1flower 10⁻⁸ M LCO application 20 ml/16 plants of 65 25 buds and 2 flower10⁻⁹ M LCO application

It was also noted that with Marigolds all the LCO treatments producedsome plants with 3 flower buds and the 50 ng treatment had some (2) with4 flower buds per plant. No untreated control plant had more than 2 budsper plant.

Trial 3: Flowering and Yield Benefit from Foliar Application of LCO

Summer Field Trial at Macdonald College Research Farm

An investigation was conducted to examine whether foliar applications ofLCO led to increased yield in tomato crop production. To determineconcentrations or strengths to be applicable, testing was conducted witha logarithmic increase in strength from 1 nanogram (ng) to 1000 ng/plantsprayed once, and on half, twice.

The trial results are presented in the following FIG. 11 and Tables 6and 7. The parameter of interest was ripened fruit which was harvested 2or 3 times a week, recording each time, both fruit weight and number offruit per set of replicates. It was known that fruit arise frompollinated flowers and that an increase in the one leads to the other.FIG. 11 records cumulative harvested (red) fruit per treatment. For thesingle LCO application it will be seen that the 10 and 100 ng/planttreatments have advanced fruiting by some 10 days over control(horizontal separation in weeks). That advance has allowed the plant tobear and ripen more fruit over the season for these treatments (see FIG.11, note height over control and Tables 6 and 7 for actual weights andnumbers harvested). Table 6 records harvested weight and numbers ofripened fruit over season and it can be seen that the average weight ofthe tomatoes is not different between treatments and control. Thus theincrease in harvested weight was due to an increase in numbersharvested, in agreement with actual enumeration. Table 7 demonstratesthat the yield increase over the season was a statistically significant17% for single application of 10 ng LCO/plant and agrees with Table 6where numbers for this application were similarly increased—some 20%.

Fruit numbers in treated plants are increased by 17%-20% arising from asimilar increase in numbers of flowers able to be pollinated.

From FIG. 11 there is a shift to earlier flowering when plants aretreated with LCOs at specific concentrations, the concentrationsrequired for physiological change being typical of a phytohormone wherea very narrow range of concentration at very low concentrations is ofbenefit-higher and lower concentrations have no effect.

Second applications are similar in trend but less clear in analysisbecause the second application led to a later increase in unharvestablegreen fruit stopped from ripening by impending frost. This would not bea problem in greenhouse operations where this indeterminate plantcontinues to yield over many months as long as root fed.

TABLE 6 LCO on Tomatoes Yield in Kg and Numbers Cumulative Yield (kg) toIncrease in Sept 23. Total No. of Tomatoes Average Wt of Tomato No. ofHarvest Total weight for 24 plants per treatment in Gms Tomatoes No. ofApplications LCO Applied LCO Applied LCO Applied LCO Applied LCO AppliedLCO Applied from 1 to 2 ng LCO per plant once Twice once Twice onceTwice applications   1 57.7200 63.8760 300 321 0.192 0.199 21  1072.2240 74.1720 363 374 0.199 0.198 11  100 65.4720 67.0000 351 3430.187 0.195 −8 1000 62.6760 71.0120 326 361 0.192 0.197 35 control -water 61.3840 65.8880 309 328 0.199 0.201 19

Conclusions 1 Treatments did not alter size of tomatoes 2 Treatmentsdetermined the number of tomatoes and therefore yield 3 All doses over10 ng LCO per plant Increased yield total number 4 Between 10 and 100 ngappears to be the best application rate 5 Double application did notImprove yields significantly over a single application

TABLE 7 LCO on Tomatoes Cumulative yield data to Sept 23 Each data paintis the average of 4 randomized rows, each of 6 plants Yield in Kg perplant Cumulative Harvest to Sept 23. Applied Applied Yield increaseYield Percent Yield Harvest once Twice over Control Increase Inc. overControl No. of applications (24 plant (24 plant Applied Applied for 2 ndApplied Applied ng LCO per plant Average) Average) once TwiceApplication once Twice   1 Not as good as Control 0.0000 — 0.00 — —  103.0093 3.0905 0.4517 0.3452 0.08 17.7% 12.6%  100 2.7280 2.7917 0.17030.0463 0.06  6.7%  1.7% 1000 2.6115 2.9588 0.0538 0.2135 0.35  2.1% 7.8% control-water 2.5577 2.7453 — — 0.19 — —

Experiment 6: Foliar Application of LCO at Variable Growth Stages

TABLE 2-1 Response Of Tomato Fruit Yield To LCO Foliar Application AtVariable Growth Stages Weight Average % of Fruit on (kg) per 24 WeightFruit # % weight vs. LCO Applied Time 24 plants plants (g/fruit) vs.control control 10 DATP* (7/4) 1276ab 150.88ab 118.2 28.63 10.8 20 DATP(7/15) 1260ab 163.78ab 130.0 27.02 20.3 30 DATP (7/25) 1199abc 160.11ab133.5 20.87 17.6 40 DATP (8/8) 1318ab 155.89ab 118.3 32.86 14.5 50 DATP(8/18) 1115bc 154.62ab 138.7 12.40 13.6 10 + 20 DATP 1125bc 153.43ab136.4 13.41 12.7 (7/4 + 7/15) 10 + 20 + 30 DATP 1282ab 161.89ab 126.329.23 18.9 (7/04 + 7/15 + 7/25) 10 + 20 + 30 + 40 DATP 1191abc 152.50ab128.0 20.06 12.0 (7/4 + 7/15 + 7/25 + 8/7) 10 + 20 + 30 + 40 + 50 DATP1373a 174.07a 126.8 38.41 27.8 (7/4 + 7/15 + 7/25 + 7/8 + 8/18)Untreated control (UC)  992c 136.15b 137.3  0  0 Significance at 5% YesYes No No No *DATP standards for Days After Transplanting

Tomato seedlings (6-leaf stage) were transplanted in farmland. Theplants were watered on the day that they were transplanted and wheneverthe soil was very dry during the season. Fertilizer (20-20-20) wasapplied at 250 kg/ha to the tomato field before transplantation. Alltomato plants were supported by sticks when they were heavily loadedwith fruit. Fruit yield (in the table) was finally cumulated at the endof the season.

Compared to the untreated control, LCO increased fruit number (up to38.4%) and total fruit weight (up to 27.8%). Five of 9 treatments hadsignificantly increased fruit number over control. For a singleapplication of LCO, the best time is 20-40 days after transplantation.Multiple applications led to increased fruit yields over a singleapplication but these results were not significantly significant.

Experiment 7: Foliar Application of LCO on Pepper

TABLE 2-2 Effect Of LCO Foliar Application On Pepper Early FruitMaturity (Sept 17) And Final Yield (Oct 9) In Horticulture Centre 2003.First 4 wk yield Treatments Fruit/plant gram/plant  14 ng/plant 5.79ab784.91ab  50 ng/plant 5.83ab 833.79ab  75 ng/plant 5.77ab 835.26ab 100ng/plant 4.87b 694.48b 141 ng/plant 6.48a 934.83a 282 ng/plant 5.02ab702.87b Water 4.87b 643.37b Significant at 5% Yes Yes

13 pepper seedlings (cv. Camelot, 6-leaf stage) were transplanted in 2rows per plot (3.5×2.5M²). The rows were covered with 65 cm width blackplastic mulch one week before transplanting. Fertilizer (10-52-10)solution of 250 ppm was applied into the planting hole through the mulchwhen transplanting (approx. 250 ml per plant). The drip irrigationsystem was set to twice a week and 4 hours each time, depending on thesoil moisture. Plants were sprayed with LCO 14 days after transplanting(5 ml/plant) and 27 days after transplanting (50 ml/plant).

LCO foliar application significantly increased fruit number in the earlystages by approximately 1 fruit per plant. Treatment of 141 ng/plant (5ml of 2×10⁻⁸M) was the best dose.

Experiment 8: Foliar Application of LCO on Corn

TABLE 2-3 Effect Of Foliar Application Of LCO On Sweet Corn Ear NumberEar Weight Marketable Average Ear Treatments Ears/ha (kg)/ha ears/haLength(cm)/ear No/plant 10⁻⁷ M once 65416.7 ab 4383.3 25000.0 13.12 c0.934 10⁻⁸ M once 67083.3 ab 4800.0 26250.0 13.24 bc 0.905 10⁻⁹ M once68333.3 a 4266.7 29166.7 13.68 abc 0.928 10⁻⁷ M twice 70833.3 a 5000.030416.7 13.81 ab 0.935 10⁻⁸ M twice 62083.3 ab 5150.0 30000.0 13.64 abc0.856 10⁻⁹ M twice 56666.7 bc 4033.3 25000.0 13.43 bc 0.886 WaterControl 49166.7 c 4416.7 39583.3 14.23 a 0.760 Significance P < 0.05 NSNS P < 0.05 NS

Fertilizer (36-12-18) was applied to the corn field at 500 kg/ha beforeseeding. A machine planter was employed to sow sweet corn grain. Plotsize was 4×4.5=18 M², and 6 rows/plot. To protect corn ears fromraccoons and other animal damage, sweet corn plants were protected withan electric fence around the plots after silking. LCO was applied once40 days after sowing or/and twice, 40 days after sowing and 58 daysafter sowing at a rate of 200 L/ha for the first application and 30 L/hafor the second application. Corn was harvested 80 days after sowing,from the two middle rows and corn ears reaching 12 cm or longer werecounted as marketable.

Foliar application of LCO in the range of 10⁻⁸-10⁻⁷M significantlyincreased total number of sweet corn ears. Total ear weight andmarketable ear number were not increased by the treatments in theseexperiments as it was necessary to harvest the crop before all werefully ripened.

Experiment 9: Effect of LCO on Grain Corn Yield

TABLE 2-4 Effect of foliar application of LCO on the fresh and dry yieldof grain corn Fresh Dry Yield Fresh Yield Dry (kg/ Yield (kg/ yieldEars/ Treatments 2 rows) (kg/ha) 2 rows) (kg/ha) 2 rows 10⁻⁷ M once 4.407333.3 3.44 5730.6 37.25ab 10⁻⁸ M once 4.57 7616.7 3.62 6040.4 37.00ab10⁻⁹ M once 4.75 7916.7 3.77 6275.6 39.00ab 10⁻⁷ M twice 5.03 8383.33.96 6606.5 40.00a 10⁻⁸ M twice 4.12 6866.7 3.23 5387.1 32.25bc 10⁻⁹ Mtwice 4.55 7583.3 3.57 5953.5 37.25ab Water Control 4.26 7100.0 3.385637.6 32.00c Significance NS NS NS NS P < 0.05

Fertilizer (36-12-18) was applied to the corn field at a rate of 500kg/ha before seeding. A machine planter was employed to plant the grain(cv. DK376, HU2650, Fludioxnil coated). To protect corn ears from birddamage, grain corn ears in the middle two rows were covered with plasticnets after silking. LCO was applied to corn plants at a rate of 200 L/hafor the first application 40 days after planting and at 400 L/ha for thesecond application 58 days after planting. The two protected middle rowsof plants were harvested by a combine 152 days after planting. Earnumber was significantly increased by all treatments of LCO applicationover untreated control, except for the 10⁻⁸M double application. Thetotal grain yield increased for all but the 10⁻⁸M double application.

Experiment 10: Effect of LCO on Ridgetwon Tomato

TABLE 2-5 Effect Of LCO Application On Ridgetwon Canning Tomato (TheFirst Harvesting Data) Increase Increase Fruit number vs. Fruit weightvs. Treatments (on 24 plants) CK (%) (kg/24 plants) CK (%) 50-0-0 206AB12.6% 11.916 9.9%  0-50-0 209AB 14.2% 12.598 16.2% 50-50-0 219A 19.7%12.567 15.9% 50-75-0 199AB 8.7% 11.421 5.3%  0-50-75 191AB 4.4% 11.3524.7% Control 183B 0 10.844 0 Significant Yes No at 5%

The experiment was conducted at Ridgetwon College, University of Guelph,Ridgetown, Ontario. Tomatoes were transplanted in single twin rows, 7 min length spaced 1.65 m apart. Treatments of LCO were applied threetimes, two weeks before flowering (28 days after transplant), two weeksafter flowering (52 days after transplant) and six weeks after flowering(69 days after transplant). Spray applications were applied using aspecialized, small plot research CO2 sprayer with a two-nozzled,hand-held boom applying 200 L/ha of spray. Rates were determined basedon 38 tomato plants per plot, replicated 4 times, equaling 152 plantsper treatment. Early fruit was harvested for yield evaluation on Aug.20, 2003. LCO foliar spray applied to tomato plants at 2 weeks beforeand after flowering significantly increased fruit number by up to 20%and also increased fruit weight by up to 16%.

Experiment 11: LCO Foliar Application in Greenhouse Tomatoes

Tomatoes were seeded and transplanted into 10″ pots 30 days later in thegreenhouse. Plants were sprayed with 5 ml (50 ng) LCO solution per plant10 days after transplant and 14 days after transplant (50 ng×2).Flowering data was collected 28 days after transplant.

LCO improved tomato early flowering, and a 50 ng/plant singleapplication better than a double application. All applications werebetter than control. See FIG. 2-1.

The same plants as FIG. 2-1 were sampled for fruit data 28 days aftertransplant.

There were no fruit seen on the control plants at this moment, however,foliar application of LCO increased tomato early fruit setting undergreenhouse conditions. Treatment of a single 50 ng LCO applicationincreased fruit set by approx. 1 fruit/plant. See FIG. 2-2.

Experiment 12: LCO Application on Marigolds

Marigolds were planted in 32-cell flat and LCO was applied foliarly toplants 4 weeks after sowing (4 flats/treatment, 1 ml/plant appliedcontaining various levels of LCO). Data collection started from thefirst flower appearing.

The higher doses of LCO (100-200 ng/plant) enhanced flowering in thefirst 2 weeks after application, whereas the lower doses (10-50ng/plant) showed better enhancement of flower 3 weeks after application.The best treatments advanced flowering by 2 days and the number offlowers at 25 days by 8%. See FIG. 2-3.

Experiment 13: LCO Application on Strawberries

Field strawberries were sprayed with a foliar application of LCO atthree dosages on the same day, as set out in FIG. 2-4. Fruit washarvested 2-3 times a week, beginning 24 days after application.

Treatment of LCO at 10-8 M (70 ng/plant) increased early fruit settingand fruit number 3-7 weeks after application from 7 to 30%.

Experiment 14: LCO Application on Cherry Tomatoes

Cherry tomato seedlings (5-week old) were transplanted into 5″ pots inthe greenhouse. LCO solutions were prepared with water and 50 ml/plantwas applied to the soil in the pot after transplantation. Ripened fruit(orange or red) were collected 8 weeks after transplantation.

LCO soil applied to transplanted cherry tomato enhanced early fruitnumber. LCO 10 ng per plant by soil application showed the best fruitenhancement at the early stage. See FIG. 2-5.

Experiment 15: LCO Application on Early Fruit Number and Yield

Red tomato seedlings (cv. Mountain Spring) were transplanted at their4-leaf stage. 7 plants in one row were transplanted in each plot of3.5×2.5M2. The row was covered with 65 cm width black plastic mulch, oneweek before transplanting. Fertilizer (10-52-10) solution of 250 ppm wasapplied into the planting hole through the mulch when transplanting(approx. 250 ml/plant). The drip irrigation system was set to twice aweek and 4 hours each time, depending on the soil moisture. Plants weresprayed with LCO 15 days after transplant (5 ml/plant) and 29 days aftertransplant (20 ml/plant). Fruits were first harvested 67 days aftertransplant.

LCO application significantly increased early fruit number and weight,but did not increase the average fruit size. The optimal application was75 ng/plant. See FIGS. 2-6 and 2-7.

Experiment 16: LCO Application on Fruit Number and Weight at End ofSeason

Tomato seedlings (6 leaf-stage) were transplanted. The plants werewatered on the day they were transplanted and whenever the soil was verydry during the season. Fertilizer (20-20-20) was applied at 250 kg/ha tothe tomato field before transplantation. All tomato plants weresupported with sticks when they were heavily loaded with fruit. Fruityield was finally cumulated at the end of the season 115 days aftertransplant.

Data showed the optimal application was 20-40 days aftertransplantation. During this period, LCO applied once at 50 ng increasedfruit number by up to 33% and fruit weight by up to %. See FIGS. 2-8 and2-9.

Experiment 17: Effect of LCO on Hot Pepper Flowering and Fruiting

30-day old seedlings were transplanted into 5″ pots and 20 days later(20 DAT) plants received the first LCO spray at 2 ml/plant (50ng/plant). The 2nd spray was conducted 3 weeks (41 DAT) after the first.Data was collected 5 weeks (55 DAT) after the first LCO application.

LCO applied in single or double applications increased early flowers upto 5% and 40% over control, respectively at 5 weeks. See FIGS. 2-10 and2-11.

30-day old seedlings were transplanted into 5″ pots and 20 days later(20 DAT) plants received the first LCO spray at 2 ml/plant (50ng/plant). The 2nd spray was conducted 3 weeks (41 DAT) after the first.Data was collected 5 weeks (55 DAT) after the first LCO application.

LCO applied in single or double applications increased the number ofearly fruits by up to 159% and 284% over control, respectively, in 5weeks. See FIG. 2-11.

Experiment 18: LCO Application on Legume

TABLE 8 Effect of LCO foliar application on grain yield and biomass ofLegumes Biomass (g/5-plant) Yield (kg/ha) Applied Applied AppliedApplied Treatments once twice once twice LCO 1 ng 49.46 AB 55.10 AB2563.96 AB 2673.54 AB LCO 10 ng 46.28 AB 60.11 A 2515.33 B 2385.75 B LCO100 ng 46.87 AB 55.09 AB 2635.00 AB 2974.17 A LCO 1000 ng 47.99 AB 47.81AB 2452.71 B 2620.42 AB Water 45.07 B 55.13 AB 2293.25 B 2421.88 BUntreated 1.1.1 N/A 1.1.2 2285.33 B control

A short heat-unit variety of soybean (cv. Nortman, HU 2425) was plantedat density of plants on the field of approx. 300 plants per plot(500,000 plants/ha). The soybean plants were first treated with LCO attheir blooming stage 24 days after planting. The treatment amounts ofLCO (detailed above) were diluted with distilled water to give a 2-litresolution sprayed over 4 plots of the treatment. The second applicationwas sprayed at the podding stage 49 days after planting. As before, theLCO was diluted with distilled water to 2-litre solution per 4 plots ofthe treatment. The plants receiving a single application were sprayedwith LCO for the first time, whereas the plants receiving a doubleapplication were sprayed for the second time. A CO₂ pressure sprayer wasemployed for this trial. The amount of fluid dispensed by the sprayerwas controlled by the nozzle size. It was calibrated with water prior tospraying with LCO. Biomass was examined 58 days after planting bydigging out by hand 5 plants per plot. The final yield was obtained byharvesting by combine the intact area (2-meter long to the end) 101 daysafter planting. The data were analyzed with the SAS program.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theclaims hereafter.

The invention claimed is:
 1. A method for initiating early fruiting in atomato plant comprising the steps of: applying to the tomato plant afirst dose of a lipo-chitooligosaccharide (LCO) after seeding in anamount from 10 ng to 100 ng, and applying to the tomato plantsubsequently a second dose of the LCO in an amount from 10 ng to 100 ng,whereby the tomato plant initiates early fruiting as compared with anuntreated tomato plant.
 2. The method of claim 1, wherein the LCO isapplied in an amount of 50 ng.
 3. The method of claim 1, wherein thesecond dose is applied 14 days after the first dose.
 4. The method ofclaim 1, wherein the LCO is applied to the foliage of the plant.
 5. Themethod of claim 1, wherein the tomato plant initiates early fruiting oneweek after the second dose.
 6. The method of claim 1, wherein the tomatoplant initiates early fruiting two weeks after the second dose.
 7. Themethod of claim 1, wherein the tomato plant initiates early fruitingthree weeks after the second dose.
 8. The method of claim 1, wherein thetomato plant initiates early fruiting four weeks after the second dose.9. A method for initiating early fruiting in a plant comprising applyingto the plant an effective amount of a lipo-chitooligosaccharide (LCO)after seeding, wherein the LCO is applied in an amount from 10 ng to 100ng, and whereby the plant initiates early fruiting as compared with anuntreated plant.
 10. The method of claim 9, wherein the plant initiatesearly fruiting one week after the LCO is applied.
 11. The method ofclaim 9, wherein the plant initiates early fruiting two weeks after theLCO is applied.
 12. The method of claim 9, wherein the plant initiatesearly fruiting three weeks after the LCO is applied.
 13. The method ofclaim 9, wherein the plant initiates early fruiting four weeks after theLCO is applied.
 14. The method of claim 9, wherein the LCO is applied inan amount of 50 ng.
 15. The method of claim 9, wherein the LCO isapplied to the foliage of the plant.
 16. The method of claim 9, whereinthe plant is a tomato plant.
 17. A method for initiating early floweringin a plant comprising applying to the plant an effective amount of alipo-chitooligosaccharide (LCO) after seeding, wherein the LCO isapplied in an amount from 10 ng to 100 ng, and whereby the plantinitiates early flowering as compared with an untreated plant.
 18. Themethod of claim 17, wherein the plant initiates early flowering one weekafter the LCO is applied.
 19. The method of claim 17, wherein the plantinitiates early flowering two weeks after the LCO is applied.
 20. Themethod of claim 17, wherein the plant initiates early flowering threeweeks after the LCO is applied.
 21. The method of claim 17, wherein theplant initiates early flowering four weeks after the LCO is applied. 22.The method of claim 17, wherein the LCO is applied in an amount of 50ng.
 23. The method of claim 17, wherein the LCO is applied to thefoliage of the plant.
 24. The method of claim 17, wherein the plant is atomato plant.